The present invention is directed to methods for blocking or delaying programmed cell death, for delivery of gene therapy to specific cells, and for treatment of cancer and other tumorgenic diseases, as well as treatment of viral infections, through the potentiation of programmed cell death in tumor or viral host cells. The present invention is also directed to assays for candidate substances which can either inhibit, or potentiate programmed cell death.
Description of the Related Art
a. Programmed Cell Death (Apoptosis)
In the last decade there has been increasing acceptance in the scientific community of the idea that cells may actually be internally programmed to die at a certain point in their life cycle. As an active cellular mechanism programmed cell death, is or apoptosis, has several important implications. First, it is clear that such an active process can provide additional means of regulating cell numbers as well as the biological activities of cells. Secondly, mutations or cellular events which potentiate apoptosis may result in premature cell death. Third, a form of cell death which is dependent on a specific active cellular mechanism can at least potentially be suppressed. Finally, an inhibition of preprogrammed cell death would be expected to lead to aberrant cell survival and could be expected to contribute to oncogenesis.
In general, apoptosis involves distinctive morphological changes including nuclear condensation and degradation of DNA to oligonucleosomal fragments. In certain circumstances it is evident that apoptosis is triggered by or is preceeded by changes in protein synthesis. Apoptosis appears to provide a very clean process for cellular destruction, in that the cells are disposed of by specific recognition and phagocytosis prior to bursting. In this manner cells can be removed from a tissue without causing damage to the surrounding cells. Thus, it can be seen that programmed cell death is crucial in a number of physiological processes, including morphological development, clonal selection in the immune system, and normal cell maturation and death in other tissue and organ systems.
It has also been demonstrated that cells can undergo apoptosis in response to environmental information. Examples include the appearance of a stimulus, such as glucocorticoid hormones for immature thymocytes, or the disappearance of a stimulus, such as interleukin-2 withdrawal from mature lymphocytes, or the removal of colony stimulating factors from hemopoietic precursors (for a review of literature see Williams, Cell, 85; 1097-1098, Jun. 28, 1991). Furthermore, it has recently been demonstrated that the response of removal to nerve growth factor from established neuronal cell cultures mimics target removal, or axiotomy, or other methods of trophic factor removal, and it has been postulated that the cellular mechanism involved in this response is a triggering of a suicide program or programmed cell death following the nerve growth factor removal. (See Johnson et al., Neurobiol. of Aging, 10: 549-552, 1989). The authors propose a xe2x80x9cdeath cascadexe2x80x9d or xe2x80x9cdeath programxe2x80x9d, which envisions that trophic factor deprivation initiates the transcription of new mRNA and the subsequent translation of that mRNA into death associated proteins which act in sequence to ultimately produce xe2x80x9ckiller proteinsxe2x80x9d. Such an intracellular mechanism seems to fit well with the characteristics of apoptosis discussed above, eg., death of specific cells without the release of harmful materials and without the disruption of tissue integrity. Furthermore, the authors indicate that inhibitors of macromolecular synthesis prevented the death of neurons in the absence of nerve growth factor.
Studies have been conducted to explore the possibility that tumor cells could be eliminated by artificially triggering apoptosis. The APO-1 monoclonal antibody can induce apoptosis in several transformed human B and T cell lines. The antibody binds to a surface protein and could act either by mimicking a positive death-inducing signal or by blocking the activity of a factor required for survival. Also, anti-FAS antibodies have similar effects, and the recent cloning and sequencing of the gene for the FAS antigen has shown that it is a 63 kilodalton transmembrane receptor. Itoh et al., Cell 66: 233-243 (1991).
However, it is important to note that neither APO-1 nor FAS can function exclusively as triggers for cell death. Both are cell surface receptors that may activate quite different responses under other circumstances. Moreover, these antigens are not confined to tumor cells and their effect on normal cells is certainly an important consideration, as is the possible appearance of variants that no longer display the antigens.
It has also been demonstrated that the cell death induced by a range of cytotoxic drugs, including several used in cancer therapy, has also been found to be a form of apoptosis. In fact, the failure of apoptosis in tumor cells could be of fundamental importance in contributing not only to the evasion of physiological controls on cell numbers, but also to resistance both to natural defenses and to clinical therapy.
It has also been demonstrated that expression of the bcl-2 gene can inhibit death by apoptosis. The bcl-2 gene was isolated from the breakpoint of the translocation between chromosomes 14 and 18 found in a high proportion of the most common human lymphomas, that being follicular B cell lymphomas. The translocation brings together the bcl-2 gene and imunoglobulin heavy chain locus, resulting in an aberrantly increased bcl-2 expression in B cells. Subsequently, Henderson et al. (Cell, 65: 1107-1115, 1991) demonstrated that expression of latent membranes protein 1 in cells infected by Epstein-Barr virus protected the infected B cells from programmed cell death by inducing expression of the bcl-2 gene. Sentman et al. (Cell, 67: 879-88, Nov. 29, 1991) demonstrated that expression of the bcl-2 gene can inhibit multiple forms of apoptosis but not negative selection in thymocytes, and Strasser et al. (Cell, 67: 889-899, Nov. 29, 1991) demonstrated that expression of a bcl-2 transgene inhibits T cell death and can perturb thymic self-censorship. Clem et al. (Science, 245: 1388-1390, Nov. 29, 1991) identified a specific baculovirus gene product as being responsible for blocking apoptosis in insect cells.
b. Herpes Virus Infections and Neurovirulence
The family of herpes virus includes animal viruses of great clinical interest because they are the causative agents of many diseases. Epstein-Barr virus has been implicated in B cell lymphoma; cytomegalovirus presents the greatest infectious threat to AIDS patients; and. Varicella Zoster Virus, is of great concern in certain parts of the world where chicken pox and shingles are serious health problems. A worldwide increase in the incidence of sexually transmitted herpes simplex (HSV) infection has occurred in the past decade, accompanied by an increase in neonatal herpes. Contact with active ulcerative lesions or asymptomatically excreting patients can result in transmission of the infectious agent. Transmission is by exposure to virus at mucosal surfaces and abraded skin, which permit the entry of virus and the initiation of viral replication in cells of the epidermis and dermis. In addition to clinically apparent lesions, latent infections may persist, in particular in sensory nerve cells. Various stimuli may cause reactivation of the HSV infection. Consequently, this is a difficult infection to eradicate. This scourge has largely gone unchecked due to the inadequacies of treatment modalities.
The known herpes viruses appear to share four significant biological properties:
1. All herpes viruses specify a large array of enzymes involved in nucleic acid metabolism (e.g., thymidine kinase, thymidylate synthetase, dUTPase, ribonucleotide reductase, etc.), DNA synthesis (e.g., DNA polymerase helicase, primase), and, possibly, processing of proteins (e.g., protein kinase), although the exact array of enzymes may vary somewhat from one herpesvirus to another.
2. Both the synthesis of viral DNAs and the assembly of capsids occur in the nucleus. In the case of some herpes viruses, it has been claimed that the virus may be de-enveloped and re-enveloped as it transits through the cytoplasm. Irrespective of the merits of these conclusions, envelopment of the capsids as it transits through the nuclear membrane is obligatory.
3. Production of infectious progeny virus is invariably accompanied by the irreversible destruction of the infected cell.
4. All herpes viruses examined to date are able to remain latent in their natural hosts. In cells harboring latent virus, viral genomes take the form of closed circular molecules, and only a small subset of viral genes is expressed.
Herpes viruses also vary greatly in their biologic properties. Some have a wide host-cell range, multiply efficiently, and rapidly destroy the cells that they infect (e.g., HSV-1, HSV-2, etc.). Others.(e.g., EBV, HRV6) have a narrow host-cell range. The multiplication of some herpes viruses (e.g., HCNV) appears to be slow. While all herpes viruses remain latent in a specific set of cells, the exact cell in which they remain latent varies from one virus to another. For example, whereas latent HSV is recovered from sensory neurons, latent EBV is recovered from B lymphocytes. Herpes viruses differ with respect to the clinical manifestations of diseases they cause.
Herpes simplex viruses 1 and 2 (HSV-1, HSV-2), are among the most common infectious agents encountered by humans (Corey and Spear, N. Eng. J. Med., 31: 686-691, 1986). These viruses cause a broad spectrum of diseases which range from mild and nuisance infections such as recurrent herpes simplex labialis, to severe and life-threatening diseases such as herpes simplex encephalitis (HSE) of older children and adults, or the disseminated infections of neonates. Clinical outcome of herpes infections is dependent upon early diagnosis and prompt initiation of antiviral therapy. However, despite some successful therapy, dermal and epidermal lesions recur, and HSV infections of neonates and infections of the brain are associated with high morbidity and mortality. Earlier diagnosis than is currently possible would improve therapeutic success. In addition, improved treatments are desperately needed.
Extrinsic assistance has been provided to infected individuals, in particular, in the form of chemicals. For example, chemical inhibition of herpes viral replication has been effected by a variety of nucleoside analogues such as acyclovir, 5-flurodeoxyuridine (FUDR), 5-iododeoxyuridine, thymine arabinoside, and the like.
Some protection has been provided in experimental animal models by polyspecific or monospecific anti-HSV antibodies, HSV-primed lymphocytes, and cloned T cells to specific viral antigens (Corey and Spear, N. Eng. J. Med., 314: 686-691, 1986). However, no satisfactory treatment has been found.
The xcex3134.5 gene of herpes simplex virus maps in the inverted repeat region of the genome flanking the L component of the virus. The discovery and characterization of the gene was reported in several articles (Chou and Roizman, J. Virol., 5: 629-635, 1986, and J. Virol., 64: 1014-1020, 1990; Ackermann et al., J. Virol., 58: 843-850, 1986). The key features are: (i) the gene encodes a protein of 263 amino acid in length; (ii) the protein contains Ala-Thr-Pro triler repeat ten times in the middle of the coding sequence; (iii) the protein is basic in nature and consists of large number of Arg and Pro amino acids; (iv) the promoter of the gene maps in the a sequence of the genome which also serves several essential viral functions for the virus; (v) the cis-acting element essential for the expression of the gene xcex3134.5 is contained within the a sequence, in particular, the DR2 (12 base pair sequence repeated 22 times) and Ub element. This type of promoter structure is unique to this gene and not shared by other viral gene promoters.
The function of the gene xcex3134.5 in its ability to enable the virus to replicate, multiply and spread in the central nervous system (CNS) was demonstrated by a set of recombinant viruses and by testing their abilities to cause fatal encephalitis in the mouse brain. The mutant viruses lacking the gene therefore lost their ability to multiply and spread in the CNS and eyes and therefore is non-pathogenic. See Chou et al., Science, 250: 1212-1266, 1990.
The xcex3134.5 gene functions by protecting the nerve cells from total protein synthesis shutoff in a manner characteristic of programmed cell death (apoptosis) in neuronal cells. The promoter appears to contain stress response elements and is transactivated by exposure to W irradiation, viral infection, and growth factor deprivation. These data suggest that the gene xcex3134.5 is transactivated in the nerve cells at times of stress to prevent apoptosis.
The significance of these findings therefore lies in the fact that xcex3134.5 extends viability or lends protection to the nerve cells so that in this instance, the virus can replicate and spread from cell to cellxe2x80x94defined as neurovirulence. It also appears that the protection can be extended to other toxic agents or environmental stresses to which the cell is subjected. An important aspect about the nature of the neurons, unlike any other cells in human, is the fact that neurons in the brain, eyes or CNS do not regenerate which forms the basis of many impaired neurological diseases. Any genes or drugs that extend the life of cells from death or degeneration can be expected to have a significant impact in the area of neural degeneration.
The role of xcex3134.5, and anti-apoptosis factors, in infected cells is in its early stages of elucidation. Recent studies have suggested that Epstein-Barr virus enhances the survival capacity of infected cells through latent membrane protein 1(LMP1)-induced up-regulation of bcl-2. In that system it is postulated that LMP 1 induced bcl-2 up regulation gives virus infected B cells the potential to by-pass physiological selection and gain direct access to long lived memory B cell pools. However, bcl-2 expression fails to suppress apoptosis in some situations, for example upon withdrawal of interleukin-2 or interleukin-6. Moreover, the intracellular mechanism of action of bcl-2 expression remains unknown.
c. Programmed Cell Death and Disease Therapy
In light of the foregoing, it is apparent that the expression of xcex3134.5 in CNS cells added an extra dimension of protection to the neurons against viral infection, and naturally ocurring and stress-induced apoptosis. An appreciation of this extra dimension of protection can be utilized in novel and innovative means for control and treatment of central nervous system (CNS) disorders. Specifically, treatment of CNS degenerative diseases, including Alzheimer""s disease, Parkinson""s disease, Lou Gerig""s disease, and others the etiology of which may be traceable to a form of apoptosis, and the treatment of which is currently very poor, could be improved significantly through the use of either the 134.5 gene in gene therapy or the protein expressed by 134.5 as a therapeutic agent. This is especially critical where the death of neuronal cells is involved, due to the fact that, as noted, such cells do not reproduce post-mitotically. Since a finite number of neurons are available it is crucial to have available methods and agents for their protection and maintenance. xcex3134.5 is also a very useful gene for assays of substances which mimic the effect of xcex3134.5 and block stress of biologically induced programmed cell death.
Furthermore, the HSV-1 virus, appropriately modified so as to be made non-pathogenic, can serve as a vehicle for delivery of gene therapy to neurons. The HSV-L virus is present in neurons of the sensory ganglia of 90% of the world""s human population. The virus ascends into neuronal cell bodies via retrograde axonal transport, reaching the axon from the site of infection by the process of neurotropism. Once in the neuronal cell body the virus remains dormant until some form of stress induces viral replication (e.g. UV exposure, infection by a second virus, surgery or axotomy). As noted, the use of HSV-1 as a vector would necessitate construction of deletion mutants to serve as safe, non-pathogenic vectors. Such a virus would act as an excellent vector for neuronal gene therapy and its use would be an especially important development since few methods of gene therapy provide a means for delivery of a gene across the central nervous system""s blood-brain barrier.
Moreover, other viruses, such as HSV-2, picornavirus, coronavirus, eunyavirus, togavirus, rahbdovirus, retrovirus or vaccinia virus, are available as vectors for xcex3134.5 gene therapy. As discussed with regard to the use of HSV-1 viruses, these vectors would also be altered in such a way as to render them non-pathogenic. In addition to the use of an appropriately mutated virus, implantation of transfected multipotent neural cell lines may also provide a means for delivery of the xcex3134.5 gene to the CNS which avoids the blood brain barrier.
In addition, use of the HSV-1 virus with a specific mutation in the xcex3134.5 gene provides a method of therapeutic treatment of tumorogenic diseases both in the CNS and in all other parts of the body. The xe2x80x9cxcex3134.5 minusxe2x80x9d virus can induce apoptosis and thereby cause the death of the host cell, but this virus cannot replicate and spread. Therefore, given the ability to target tumors within the CNS, the xcex3134.5 minus virus has proven a powerful therapeutic agent for hitherto virtually untreatable forms of CNS cancer. Furthermore, use of substances, other than a virus, which inhibit or block expression of genes with anti-apoptotic effects in target tumor cells can also serve as a significant development in tumor therapy and in the treatment of herpes virus infection, as well as treatment of infection by other viruses whose neurovirulence is dependent upon an interference with the host cells"" programmed cell death mechanisms.
This invention relates to methods for the prevention or treatment of programmed cell death, or apoptosis, in neuronal cells for therapy in connection with neurodegenerative diseases, as well as methods of treatment of cancer and other tumorogenic diseases and herpes virus infection. The present invention also relates to assay methodologies allowing for the identification of substances capable of modulating the effects of the xcex3134.5 gene or its protein expression product ICP34.5, i.e., substances capable of potentiating or inhibiting their effects. Additionally, the present invention also relates to assay methodologies designed to identify candidate substances able to mimic either xcex3134.5 expression or the activity of ICP34.5. The present invention also relates to methods of delivering genes to cells for gene therapy.
In one illustrative embodiment of the present invention a method of preventing or treating programmed cell death in neuronal cells is described in which a non-pathogenic vector is prepared which contains the xcex3134.5 gene. This vector is then introduced into neuronal cells which are presently undergoing or are likely to undergo programmed cell death. Those skilled in the art will realize that several vectors are suitable for use in this method, although the present invention envisions the use of certain unique and novel vectors designed specifically for use in connection with delivery of the xcex3134.5 gene.
One such vector envisioned by the present invention is the HSV-1 virus itself, modified so as to render it non-pathogenic. Because of the unique capability of the HSV-1 virus to use an axon""s internal transport system to move from the peripheral nerve endings of the neuron into the neuronal cell body, the present invention proposes the use of the non-pathogenic HSV-1 virus injected into the vicinity of the synaptic terminals of affected neurons, or in the area of a peripheral wound or lesion or other appropriate peripheral locus. The HSV-1 virus containing the xcex3134.5 gene, under a different target-specific promoter, would then be transported into the neuronal cell body via retrograde axonal transport.
The present invention envisions specific genomic modifications being introduced into the HSV-1 virus in order to render the virus non-cytotoxic. These modifications could include deletions from the genome, rearrangements of specific genomic sequences, or other specific mutations. One example of such a modification comprises modification or deletion of the xcex14 gene which encodes the ICP4 protein. Deletion or modification of the gene expressing ICP4 renders the HSV-1 virus unable to express genes required for viral DNA and structural protein synthesis. However, the xcex3134.5.gene placed under a suitable promoter would be expressed, thus inducing an anti-apoptotic effect in the neuron without the potential for stress induced neurovirulence. Other genes which might be modified include the the xcex30 gene. The present invention also envisions the use of other vectors including, for example, retrovirus, picorna virus, vaccinia virus, HSV-2, coronavirus, eunyavirus, togavirus or rhabdovirus vectors. Again, use of of such viruses as vectors will necessitate construction of deletion mutations so that the vectors will be safe and non-pathogenic.
Another method by which the present invention envisions introducing the xcex3134.5 gene into neuronal cells undergoing or likely to undergo programmed cell death, is through the use of multi-potent neural cell lines. Such lines have been shown to change phenotype in vitro and have also been demonstrated to become integrated into the central nervous system of mice and to differentiate into neurons or glia in a manner appropriate to their site of engraftment. Snyder, et al., Cell, 68: 33-51, 1992. Transplant or engraftment of multi-potent neural cell lines into which the xcex3134.5 gene has been introduced into an area of the central nervous system in which cells are undergoing or are likely to undergo programmed cells death is expected to lead to reversal and inhibition of programed cell death.
It is expected that the ability of xcex3134.5 to inhibit apoptosis will be a boon not only in human medicine, but also in basic scientific research. In this regard the present invention also envisions the use of the xcex3134.5 gene in the extension of the life of neuronal cells in cell culture. Introduction of a non-cytotoxic vector into cultured neuronal cells will have an anti-apoptotic effect and will thereby extend the life of cell cultures. This in turn will extend the time periods over which experimentation may be conducted, and can also be expected to decrease the cost of conducting basic research.
In addition to utilizing a vector comprising the xcex3134.5 gene, the present invention also discloses a method of preventing or treating programmed cell death in neuronal cells which involves the use of the product of expression of the xcex3134.5 gene. The protein expressed by xcex3134.5 is called ICP34.5. Ackermann, et al. (J. Virol., 58: 843-850, 1986) reported that ICP34.5 has an apparent molecular weight of 43,500 upon SDS-polyacrylamide gel electrophoresis, appears to accumulate largely in the cytoplasm of HIV infected cells, and in contrast to many HSV-1 proteins, ICP34.5 has been demonstrated to be soluble in physiologic solutions.
In practicing this method or the method in which the xcex3134.5 gene is introduced into cells, it is envisioned that the xcex3134.5 gene or a biological functional equivalent thereof could be used for gene therapy, or ICP34.5 in a purified form or a biological functional equivalent of the ICP34.5 protein could be utilized as an anti-apoptotic agent. As used herein, functional equivalents are intended to refer to those proteins, and their encoding nucleic acid sequences, in which certain structural changes have been made but which nonetheless are, or encode, proteins evidencing an effect similar to that of ICP34.5.
In light of the fact that certain amino acids may be substituted for other amino acids in a protein without appreciable loss of defined functional activity, it is contemplated by the inventors that various changes may be made in the sequence of the ICP34.5 protein (or the underlying DNA of the xcex3134.5 gene) without an appreciable loss of biological utility or activity. Amino acids with similar hydropathic scores may be substituted for one another (see Kyte et al., J. Mol. Biol., 157: 105-132, 1982, incorporated herein by reference), as may amino acids with similar hydrophilicity values, as described in U.S. Pat. No. 4,554,101, incorporated herein by reference.
Therefore, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
This embodiment of the present invention describes a method which involves combining ICP34.5 or a biological functional equivalent thereof with a pharmaceutically acceptable carrier in order to form a pharmaceutical composition. (It should be understood in subsequent discussions that when xcex3134.5 or ICP34.5 are referred to, the inventors intend to include biological functional equivalents, including any chemicals which mimic the effect of xcex3134.5.) Such a composition would then be administered to neurons likely to undergo or undergoing programmed cell death. Such a composition could be administered to an animal using intravenous, intraspinal injection or, in certain circumstances, oral, intracerebral or intraventricular administration may be appropriate. Furthermore, neuronal cells in culture could also benefit from administration of ICP34.5 through administration directly into the medium in which the neuronal cells are grown.
ICP34.5 can be prepared using a nucleic acid segment which is capable of encoding ICP34.5 (i.e., the xcex3134.5 gene or a biological functional equivalent). Such a segment could be expressed using, for example, a technique involving transferring the xcex3134.5 segment into a host cell, culturing the host cell under conditions suitable for expression of the segment, allowing expression to occur, and thereafter isolating and purifying the protein using well established protein purification techniques. The nucleic acid segment would be transferred into host cells by transfection or by transformation of a recombinant vector into the host cell.
A particularly important embodiment of the present invention relates to assays for candidate substances which can either mimic the effects of the xcex3134.5 gene, or mimic the effects of ICP34.5, as well as assays for candidate substances able to potentiate the function of xcex3134.5 or potentiate the protective function of ICP34.5. Additionally, methods for assaying for candidate substances able to inhibit either xcex3134.5 expression or the activity of ICP34.5 are also embodiments of the present invention.
In an exemplary embodiment, an assay testing for candidate substances which would block the expression of the anti-apoptosis gene or inhibit the activity of an anti-apoptotic protein such as ICP34.5 would proceed along the following lines. A test plasmid construct bearing the a sequence promoter and portions of the coding sequence of xcex3134.5 is fused to the lacZ reporter gene, or any other readily assayable reporter gene. This construct is then introduced into an appropriate cell line, for example a neuroblastoma or PC12 cell line, by G418 selection. A clonal and continuous cell line for screening purposes is then established. A control plasma construct bearing an HSV late promoter (a promoter, which would normally not be expressed in cell lines and not induced to express by a stress factor which would normally induce apoptosis) is fused to the same or different indicator gene. This construct is also introduced into a continuous clonal cell line and serves as a control for the test cell line. The anti-apoptosis drugs would then be applied. Environmental stresses which typically trigger a sequence promoter activation and cause programmed cell death, such as UV injury, viral infection or deprivation of nerve growth factor, would then be applied to the cells. In control cells, the stress should have no effect on the cells and produce no detectible reaction in the assay. Stress in a test cell line in the absence of a positive candidate substance would give rise to an appropriate reaction, typically a colorimetric reaction. Introduction of stress to the test cell line in the presence of the candidate substance would give rise to an opposite colorimetric reaction indicating that the candidate substance interferes either with expression of the xcex3134.5 gene, or with the ability of the substance to interfere with the anti-apoptotic activity of ICP34.5.
Similarly, the present invention describes an assay for candidate substances which would mimic or potentiate the activity of ICP34.5, or which would mimic the expression of xcex3134.5, and such an assay would proceed along lines similar to those described above. A test cell line (e.g., a neuroblastoma cell line) constitutively expressing ICP34.5 and a fluorescent tagged cellular gene or any other tag providing an easily detected marker signalling viability of the cells is produced. In addition, a corresponding null cell line consisting of an appropriate indicator gene, for example the a-lacZ indicator gene, and the same host indicator gene as in the test cell line is also produced. Also, a third cell line (e.g., a vero cell line) consisting of the same indicator gene and the identical host indicator gene is also produced. Again, environmental stresses which trigger programmed cell death in the absence of xcex3134.5 are applied to the cells. Candidate substances are also applied in order to determine whether they are able to mimic or. potentiate the anti-apoptotic effects of xcex3134.5 expression or the anti-apoptotic activity of ICP34.5 or biological functional equivalents thereof.
The present invention also embodies a method of delivering genes for gene therapy. In an exemplary embodiment, the method involves combining the gene used for gene therapy with a mutated virus such as those described above, or with the HSV-1 virus rendered non-pathogenic. The gene and the virus are then combined with a pharmacologically acceptable carrier in order to form a pharmaceutical composition. This pharmaceutical composition is then administered in such a way that the mutated virus containing the gene for therapy, or the HSV-1 wild type virus containing the gene, can be incorporated into cells at an appropriate area. For example, when using the HSV-1 virus, the composition could be administered in an area where synaptic terminals are located so that the virus can be taken up into the terminals and transported in a retrograde manner up the axon into the axonal cell bodies via retrograde axonal transport. Clearly, such a method would only be appropriate when cells in the peripheral or central nervous system were the target of the gene therapy.
The present invention also envisions methods and compositions for the treatment of cancer and other tumorogenic diseases, as well as herpes infections or other infections involving viruses whose virulence is dependent upon an anti-apoptotic effect. Candidate substances identified as having an inhibiting effect upon either the expression or activity of ICP34.5 identified in the assay methods discussed above could be used to induce cell death in target tumor cells, or in virus-infected cells. Pharmaceutical compositions containing such substances can be introduced using intrathecal, intravenous, or direct injection into the tumor or the infected area, as appropriate.